Journal: eLife

Article Title: Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart

doi: 10.7554/elife.50163

Figure Lengend Snippet: Figure 6. Nrg1/ErbB2 signaling induces glycolysis genes in zebrafish and mouse. (a) Cartoon showing experimental procedure to induce cardiomyocyte specific Nrg1 expression in zebrafish. Panels show in situ hybridization for hexokinase 1 (hk1) and lactate dehydrogenase a (ldha) expression on sections of control hearts (b-act2:BSNrg1) and Nrg1 OE hearts (cmlc2:CreER; b-act2:BSNrg1). Scale bars represent 100 mm. (b) qPCR results for glycolytic genes showing their relative fold change in DMSO treated (n = 9) (blue) and AG1478 treated (n = 9) (green) nppa:mCitrine high border zone cardiomyocytes at 3dpi compared to uninjured adult cardiomyocytes (n = 4) (yellow). Error bars represent standard deviation. (c) Upper panel: Cartoon showing the experimental procedure to analyse metabolic gene expression after activating ErbB2 signaling in the murine heart. Lower panel: qPCR results for metabolic genes showing their relative fold change in caErbB2 OE (n = 4) heart compared to control WT hearts (n = 4). Error bars represent standard deviation. (d) Immunohistochemistry for phospho-PDH3 on sections of control and caErbB2 OE hearts. Scale bars represent 100 mm. *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. The online version of this article includes the following source data and figure supplement(s) for figure 6:

Article Snippet: ADULT ZEBRAFISH: nppa:mCitrine zebrafish were cryoinjured and received two overnight pulses of DMSO (1:2000) or 5 mM AG1478 (10 mM stock in DMSO; Selleck Chemical, Houston, TX) from 1dpi to 2dpi and from 2dpi to 3dpi.

Techniques: Expressing, In Situ Hybridization, Control, Standard Deviation, Gene Expression, Immunohistochemistry

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 1 Autophagy stimulation by ABT737. (a, b) Levels of autophagy induced by ABT737 in U2OS cells stably expressing GFP- LC3 were measured in time-dependent manner as described in Materials and methods section. Representative microphotographs of cells (size bar 50 mm) cultured in the presence of ABT737 (1 mM) are shown in (a) and the number of GFP-LC3 puncta per cell (means±s.e.m., n ¼ 3 separate experiments) are quantified in (b). (c, d) Effect of Bafilomycin A1 (Baf A1) on ABT737-induced autophagic vacuolization. GFP-LC3-expressing HCT116 cells were treated with Baf A1 (1 nM) for further 24 h. Panel c depicts the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) (means±s.e.m., n ¼ 3). (d) Representative immunoblots (n ¼ 3) of HCT116 cells treated with ABT737 and BafA1 for the indicated time. (e) Involvement of Vps34 in ABT737- induced autophagy. HeLa cells were transfected with a control siRNA or a siRNA specific for Vps34. After 48 h, the cells were treated for 12 h with ABT737 (1 mM). Each experiment has been repeated at least three times, yielding similar results. (d, e) Actin levels were assessed to ensure equal loading of lanes.

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Transfection, Control

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 2 Implication of AMPK, mTOR and IKK in the autophagy induction by ABT737. Immunoblot detection of the levels of p62, LC3I/II, p53 and Sirtuin1, as well as the phosphorylation status of AMPKa (Y172), ACCa (S79), mTOR (S2448), p70S6K (Y421/S424), IkBa (S32/36), IKKa/b (S176 or S180, respectively) and NEMO (S376) in different human cell lines (a–c) stimulated with ABT737 (1 mM) for the indicated time. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading. Data are representative of three independent experiments.

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Western Blot, Phospho-proteomics

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 3 Involvement of AMPKa and mTOR in ABT737-induced autophagy. (a) Dose-dependent induction of autophagy by ABT737. Representative immunoblots of LC3, p62 and phosphorylated AMPKa (Y172) or ACCa (S79) were performed on lysates from HCT116 cells that were treated with ABT737 at the indicated concentrations for 12 h. (b) Immunoblot detection of LC3 lipidation and p62 degradation in HCT116 depleted from AMPKa. At 48 h after the transfection with the indicated siRNAs, the cells were treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the protein is also shown. Actin levels were assessed to ensure equal loading of lanes. Data in (a) and (b) are representative of three independent experiments. (c) Implication of mTOR in ABT737-induced autophagy. HeLa cells were simultaneously transfected with GFP-LC3 and equivalent amounts of plasmids coding for control vector, Rheb D60K (dominant-negative construct) or Rheb Q64L (constitutively active construct). After 24 h, the cells were treated with ABT737 (1 mM) for 12 h. The percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is shown (means±s.e.m., three independent experiments).

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Dominant Negative Mutation, Construct

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 4 Involvement of IKK in ABT737-induced autophagy. (a, b) Pharmacological inhibition of IKK pathway reduces autophagy increased by ABT737. (a) LC3 maturation and p62 degradation in HCT116 treated with ABT737 (1 mM) in the presence of Bay11-7082 (1 mM), GA (geldanamycin; 2 mM), 17-AAG (17-allylamino-geldanamycin; 2 mM) for 12 h. Actin levels were assessed to ensure equal loading of lanes. Data are representative of three independent experiments. (b) GFP-LC3-expressing HeLa, U2OS, HCT116 cells were treated under the same conditions of (a) and the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is shown (means±s.e.m., n ¼ 3). (c–e) Immunoblot detection of LC3 lipidation, as well as p62 degradation in HCT116 subjected to knockdown of IKKa, IKKb, NEMO. At 48 h after the transfection, the cells were treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the protein is also shown. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading (n ¼ 4). (f) HCT116 were transfected with unrelated (UNR) or a siRNA specific for the IKK complex subunits and subsequently (after 24 h) with a plasmid for the expression of GFP-LC3, followed by culture in the presence of ABT737 (1 mM) for 12 h (means±s.e.m., n ¼ 3). (g) Immunoblot detection of phosphorylation status of IkBa, IKKa/b and NEMO. HCT116 cells were transfected with UNR or siRNAs specific for Atg5 and Beclin 1 as mentioned previously and treated with ABT737 (1 mM) for 12 h. The efficiency of siRNA-mediated downregulation of the proteins is also shown. Actin levels were assessed to ensure equal loading of lanes. Data are representative of three independent experiments.

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Inhibition, Expressing, Western Blot, Knockdown, Transfection, Plasmid Preparation, Phospho-proteomics

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 5 Involvement of p53 and SIRT1 in ABT737-induced autophagy. (a, b) Levels of LC3I/II and p62 expression in HCT116 treated with ABT737 (1 mM) for 12 h in the presence or absence of pharmacological inhibitors of MDM2 (RITA or Nutlin-3, both used at 10 mM), proteasome (MG132, 10 mM) and SIRT1 (EX527, 100 mM) (n ¼ 3). Actin and glyceraldehyde-3-phosphate dehydrogenase levels were assessed to ensure equal loading of lanes. (c) GFP-LC3-expressing HeLa, U2OS or HCT116 cells were treated under the same conditions as in (a, b) and the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (means±s.e.m., n ¼ 3). (d, e) Immunoblot detection of LC3 lipidation, as well as p62 degradation in HCT116 subjected to knockdown of SIRT1, HDM2 and p53. At 48 h after the transfection, the cells were treated with ABT737 for 12 h. The efficiency of siRNA- mediated downregulation of the proteins is also demonstrated. Glyceraldehyde-3-phosphate dehydrogenase levels were monitored to ensure equal loading (n ¼ 5). (f) Quantification of the percentage of GFP-LC3vac in HCT116 transfected with siRNAs specific for SIRT1, HDM2 and p53, as described in Materials and methods section, and stimulated for 12 h with ABT737 (1 mM) (means±s.e.m., n ¼ 3).

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Expressing, Western Blot, Knockdown, Transfection

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 6 Pro-autophagic activity of the BH3 mimetic HA14-1. (a, b) U2OS cells stably expressing GFP-LC3 were treated with HA14- 1 and the kinetics of puncta formation was determined by videomicroscopy (means±s.e.m., n ¼ 3 separate experiments). (c) Alternatively, HeLa cells were cultured in the presence of HA14-1 (20 mM) at the time points mentioned, and the lipidation of LC3 and the expression of p62 were monitored by immunoblot. Actin levels were assessed to ensure equal loading of lanes. Results are representative of three independent determinations. (d) WT and Bax/Bak/ MEF were cultured in the presence of ABT737 (1 mM) or HA14-1 (20 mM) overnight, and the lipidation of LC3 and the expression of p62 were monitored by immunoblot. Actin levels were assessed to ensure equal loading of lanes. Results are representative of three independent determinations. (e) In the same conditions as shown in d, the percentage of cells exhibiting the accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (means±s.e.m., n ¼ 3).

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Activity Assay, Stable Transfection, Expressing, Cell Culture, Western Blot

Journal: Oncogene

Article Title: BH3 mimetics activate multiple pro-autophagic pathways.

doi: 10.1038/onc.2011.104

Figure Lengend Snippet: Figure 7 Profile of phospho-proteome phosphorylation status elicited in HeLa cells after ABT737 treatment. HeLa cells were treated with ABT737 (1 mM) for 2, 6 or 12 h. Cell extracts were then probed on human phosphoprotein arrays following manufacturer’s instructions (see Materials and methods section). (a) Representative pictures of proteome profiler array. (b) Clustering analysis for the effect of BH3 mimetic on protein kinase phosphorylation. (c, d) Representative immunoblots of selected kinases whose phosphorylation status was affected by ABT737 (1 mM) or HA14-1 (20 mM) treatment (Pyk2, Akt, STAT3 and p53) validating the phosphoprotein arrays (n ¼ 3). (e) Comparison of the effects of ABT737 (1 mM) or HA14-1 (20 mM) on the mTOR, ACC, AMPK and p70S6K activation status (n ¼ 3). (f) Effect of Vps34 and Beclin 1 downregulation on the ABT737-mediated activation of AMPKa and inhibition of mTOR, as determined by immunoblot experiments (n ¼ 3). (c–f) Actin levels were monitored to ensure equal loading (n ¼ 3).

Article Snippet: Cells were seeded in 6- or 12-well plates and grown for 24 h before treatment with ABT737 (2000, 1000, 100, 10 or 1 nM; Selleck Chemicals, Houston, TX, USA), Bay11-7082 (1 mM; BioMol Research Laboratories, Lausen, Switzerland), EX527 (100mM; Tocris Bioscience, Bristol, UK), bafilomycin A1 (BafA1; 1 nM), geldanamycin (2 mM), cisPt (10 and 20mM), 17-allylamino-geldanamycin (2 mM), MG132 (10 mM; SigmaAldrich, St Louis, MO, USA), HA14-1 (1, 5, 10 and 20mM), Nutlin-3 (10 mM) or RITA (10mM; Alexis Biochemicals, Lausen, Switzerland) for the indicated period (12 h, unless otherwise stated).

Techniques: Phospho-proteomics, Western Blot, Comparison, Activation Assay, Inhibition